Anti-carbamylated protein antibodies and the risk for arthritis

ABSTRACT

Antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the diagnosis and possibly pathogenesis in arthritis. Described are means and methods for determining antibodies against homocitrulline-containing proteins or carbamylated proteins/peptides (anti-CarP) for the classification of individuals suffering from, or at risk of suffering from, arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/NL2012/050056, filed Feb. 1, 2012, designating the United States of America and published in English as International Patent Publication WO 2012/105838 A1 on Aug. 9, 2012, which claims the benefit under Article 8 of the Patent Cooperation Treaty and under 35 U.S.C. §119(e) to European Patent Application Serial No. 11182399.3, filed Sep. 22, 2011, and to European Patent Application Serial No. 11153046.5, filed Feb. 2, 2011.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) OR (e)—SEQUENCE LISTING SUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT version and a PDF version of the Sequence Listing have been submitted concomitant with this application, the contents of which are hereby incorporated by reference.

TECHNICAL FIELD

The disclosure relates to the fields of post-translational modification and arthritis. In particular, it relates to methods classifying samples of individuals based on the detection of post-translationally modified proteins or peptides or antibodies specific for post-translationally modified proteins or peptides in a sample containing a body fluid of the individual.

BACKGROUND

There are over 100 different forms of arthritis. The most common form is osteoarthritis (degenerative joint disease). Osteoarthritis is most commonly the result of a trauma or of an infection of the joint, albeit that there are also not readily identifiable causes. The latter are often collectively referred to as “age-related osteoarthritis.” Other forms of arthritis are, for example, rheumatoid arthritis, psoriatic arthritis, and related autoimmune diseases.

The major complaint by individuals who have arthritis is joint pain. Pain is often constant and may be localized to the joint affected. The pain from arthritis is often the result of damage to the joint or the result of the inflammation that occurs around the joint. Other complaints are pain as a result of muscle strains caused by forceful movements against stiff, painful joints and fatigue.

The diagnosis of patients with joint-associated pain is not easy as the complaints are typically vague and can be attributed to a variety of different causes, some of which are not arthritis. Indeed, many patients that first see a doctor with joint pain-associated complaints go into remission and don't develop a chronic form of arthritis, whereas a significant minority progress to develop Rheumatoid Arthritis (RA). It is clear that individuals that go into remission spontaneously do not need to receive treatment, whereas individuals that progress would benefit considerably from early treatment. To discriminate between the respective groups, the field has developed a series of tests with which the diagnosis of arthritis can be made with more certainty. Such tests presently involve the screening of tissue samples and/or body fluid samples for the presence of arthritis indicators therein. Such indicators are, among others, determination of “chronic” inflammation indicators such as, for instance, certain chemokines, cytokines and other immune cell signaling factors; the determination of “accumulation of” active immune cells in joints, and/or the presence of the certain factors in the blood, the most notable of which is rheumatoid factor. Recently, tests directed toward the detection of citrullinated protein or peptides or antibodies specific for such citrullinated protein or peptide have been developed as a useful tool for such tests. The availability of such tests has greatly improved the diagnosis of individuals suspected of having a form of arthritis. These tests also aid the clinician in giving a more accurate prognosis for the future development of the disease to individuals suffering from arthritis. However, in spite of these developments, the diagnosis of arthritis or individuals at risk thereof still leaves much to be desired.

For example, in the Netherlands, the recommendation to diagnose RA is based on a probability score generated by the ACR/EULAR 2010 criteria. This criteria combines clinical features such as involvement of type and number of joints, presence or absence of the serological factors, rheumatoid factor and anti-CCP antibodies, presence or absence of acute phase proteins such as CRP, and duration of complaints. Patients that are diagnosed with RA according to this protocol need to have more than six points. The fact that only the clinical involvement and duration of complaints are sufficient for a positive diagnosis indicates that the arthritis population is very heterogeneous and, in fact, often a wrong diagnosis is made.

In the Netherlands and in other countries, tests for the typing of arthritis or diagnoses of an individual at risk of developing arthritis presently include tests for the detection of antibodies specific for citrullinated protein or peptide in samples of body fluids of such patients. These tests have led to the insight that arthritis patients can be classified on the basis of positivity for antibodies directed toward citrullinated proteins (ACPA). The identification of ACPA has had an important impact on the understanding of RA.⁽¹⁾ Major differences have been observed when comparing ACPA-positive vs. ACPA-negative RA patients regarding genetic and environmental risk factors,⁽²⁾ progression,⁽³⁾ remission,⁽⁴⁾ and response to treatment.⁽⁵⁾ Over the recent years, much more insight has been gained into the occurrence and etiopathology of ACPA-positive RA. However, much less information is available on ACPA-negative RA. In part, this is because it is relatively difficult to identify or even subgroup these individuals as no good assays are currently available. Interestingly, rituximab treatment has been reported to also be beneficial in patients negative for RF and ACPA.^((6, 7))

The post-translational modification of arginine residues into citrulline residues by the PAD enzymes is the essential step to generate antigens for ACPA.⁽¹⁾ Under physiological circumstances, this citrullination is important in tissues like hair and skin to generate layers of tissue that are not-well connected.⁽⁸⁾ Also, in the nucleus, citrullination plays a role in epigenetic regulation⁽⁹⁾ and condensation of chromatin, which is important, both in translation⁽⁸⁾ and in host defense against pathogens.⁽¹⁰⁾ Under pathological conditions where cell death may overwhelm the phagocytic capacity, necrotic cell death may release PAD into the extracellular space, where higher calcium concentrations now also allow other host molecules to become citrullinated.⁽⁸⁾ Since many of these molecules will be presented to the immune system as non-self, it can induce an antibody response in some individuals. Citrulline highly resembles (FIG. 1) another post-translationally modified amino acid called homocitrulline.⁽¹¹⁾ Homocitrulline is only one carbon longer, but similar in structure.⁽¹¹⁾ Homocitrulline is generated from a Lysine residue following an attack from cyanate, which exists in the body in equilibrium with urea. Under physiological conditions, the urea concentration may be too low to allow extensive carbamylation (the process of changing lysine to homocitrulline). In conditions of renal failure, the urea concentration increases and carbamylation can be readily detected. However, most carbamylation is taking place during inflammation when myeloperoxidase (MPO) is released from neutrophils.⁽¹²⁾ This enzyme strongly shifts the equilibrium of urea toward cyanate, now allowing more carbamylation to occur.⁽¹³⁾ It has been shown recently that homocitrulline-containing proteins are present in the RA joint and that this may affect T-cell triggering and autoantibody formation in animal models.^((11, 14)) Although highly similar, carbamylation differs from citrullination as, next to their structural difference, lysine and not arginine is modified. Therefore, homocitrulline will, by definition, be located at other positions in proteins than citrulline.

DISCLOSURE

Autoantibodies against carbamylated proteins have been found to be present in arthritis, and it has been determined that measurement of such antibodies is useful in the diagnosis, prognosis, and management (e.g., treatment) of early arthritis and RA. Thus, provided is a method for classifying an individual that is suffering from, or at risk of suffering from, a faun of arthritis, the method comprising determining whether a sample comprising a body fluid of the individual comprises an anti-Carbamylated Protein (anti-CarP) antibody.

An individual that is suffering from arthritis can be classified, on the basis of anti-CarP antibodies, as low or high risk of developing a more severe form of arthritis. Individuals with arthritis that are positive for anti-CarP tend to develop a more severe form of arthritis than anti-CarP-negative individuals with arthritis, at least within any given time period. The anti-CarP-positive individuals also tend to progress into more severe forms more quickly when compared with anti-CarP-negative individuals. The method is thus preferably used to allocate an individual that is suffering from a form of arthritis to a group with a lower or a higher than average risk for progressing to a more severe or chronic form of arthritis. In a preferred embodiment, the individual is suffering from undifferentiated arthritis at presentation. In a preferred aspect of this embodiment, the more severe or chronic form is RA. In another preferred aspect of this embodiment, the more severe or chronic form is juvenile arthritis, preferably juvenile idiopathic arthritis. In a preferred embodiment, the group with a lower than average risk is a group that has a higher than average incidence of spontaneous remission of the arthritis complaints.

The at-risk population may be healthy individuals, patients suffering from undifferentiated arthritis or arthralgia, autoantibody-positive individuals with joint complaints, autoantibody-positive individuals, or family members from patients with arthritis. “Juvenile idiopathic arthritis” is the term used for a subset of arthritis seen in childhood, which may be transient and self-limiting or chronic. Children with juvenile idiopathic arthritis are considered an at-risk population, as some of these patients may develop a chronic form of arthritis.

The application shows that detection of anti-CarP antibodies is useful in individuals that present with undifferentiated arthritis, arthralgia, and other joint complaints, and/or with juvenile arthritis. The presence or absence of anti-CarP antibodies is predictive of the development of RA or persistent arthritis later in life. This predictive power is observed both in ACPA-negative and ACPA-positive subjects.

The method may be used to predict whether the individual is at risk of developing RA or persistent arthritis later in life.

For instance, human carbamylated fibrinogen can be used as a target for anti-CarP antibodies. Carbamylated fibrinogen is recognized by both IgG and IgA anti-CarP antibodies. Intact fibrinogen, or any of peptides derived from carbamylated fibrinogen, can be used as targets in assays to detect anti-CarP antibodies. The intact protein or any of the peptides derived from fibrinogen can be used directly in the assay or can be immobilized via an attached biotin group that will bind to streptavidin-coated surfaces or can be used in any other way to detect anti-CarP antibodies. In a preferred embodiment, a method according to the invention is provided, wherein the anti-CarP antibody is capable of specifically binding carbamylated fibrinogen. Throughout the description, the abbreviation “anti-Ca-Fib antibody” is used to indicate such antibody capable of specifically binding carbamylated fibrinogen.

Progression of disease in arthritis, for instance, progressive pain complaints or progressive joint damage, is not a constant. The progression may be faster or slower for some period of time. When mention is made herein of a lower or higher risk of progression, this is typically compared to the average risk of progression within the group of individuals that is studied.

Progression of diseases is at a group level established every year, so that follow-up data can be analyzed using repeated measurement analysis. Yet, at the individual levels, positivity for anti-CarP, preferably anti-Ca-Fib, is predictive for future progression. Currently, several methods including MRI, ultrasound and other techniques are available to measure progression of disease in the short term.

To assess whether a sample comprises anti-CarP antibodies, preferably anti-Ca-Fib antibodies, a test for the presence of the antibodies is performed. Such tests can involve, but are not limited to, ELISA and/or Western blot, using one or several carbamylated proteins and/or peptides. Commonly, though not necessarily, the result obtained for the sample is compared with a reference. The reference is typically the result of a similar test, preferably the same test, performed on one or a number of healthy individuals (i.e., not known to suffer from arthritis and not known to be at immediate risk of developing arthritis). The result of the sample can be directly compared with the result of the reference, or the reference can be used to determine a threshold, below which any sample is to be judged a negative for anti-CarP, preferably anti-Ca-Fib, antibodies when below the threshold or positive when above the threshold.

Further provided is a method for providing a prognosis for the development of arthritis to an individual suffering from arthritis, the method comprising determining whether a sample comprising a body fluid of the individual comprises an anti-Carbamylated Protein (anti-CarP) antibody, and estimating the future severity of the arthritis based on the detection of the anti-CarP antibody in the sample. In a preferred embodiment, the anti-CarP antibody is capable of specifically binding carbamylated fibrinogen (anti-Ca-Fib antibody). This estimation is typically accompanied by a time interval within which the more severe form or progression becomes apparent or not (see hereinabove).

One advantage of the classifications as indicated hereinbefore is that the groups of individuals have more homogeneous genetic profile within each group than with other methods. Another advantage is that the groups of individuals are more homogeneous in their response to (prophylactic) treatment. Since it has been shown that early aggressive treatment is beneficial^((18, 19)) provided are methods for arthritis treatment of individuals suffering from, or at risk of suffering from, arthritis, the method comprising an arthritis diagnosis of the individual wherein the diagnosis comprises a method for determining an anti-CarP antibody, preferably an anti-Ca-Fib antibody, in a sample comprising a body fluid of the individual. Preferably, the sample was determined to contain an anti-CarP antibody, preferably an anti-Ca-Fib antibody. A more stringent treatment of the anti-CarP or anti-Ca-Fib-positive individual is beneficial to the patient. The treatment is typically a therapeutic treatment given to a patient that was diagnosed with arthritis prior to receiving the treatment. Further provided is a method of treating an individual suffering from arthritis with an arthritis medication and/or treatment, the method characterized in that the individual was diagnosed with a method hereof prior to the treatment. Preferably, the individual was diagnosed as suffering from arthritis with a method for classifying individuals suffering from arthritis of the invention, prior to receiving the arthritis medication and/or treatment. Further provided is a method for the prophylactic treatment of an individual at risk of developing arthritis with an arthritis medication and/or treatment, the method characterized in that the individual was diagnosed being at risk of developing arthritis with a method for classifying individuals of the invention, prior to receiving the arthritis medication and/or treatment. Treatment, in this case, prophylactic, can also be given to individuals that are classified to be at risk of developing the disease in the near future, typically within one year of classification. Such prophylactic treatments are capable of at least postponing the onset of the disease and/or reducing the severity of the disease.

The methods used to classify arthritis patients or individuals at risk of developing arthritis typically involve a number of different tests. Such tests may also be combined with a method of the invention to arrive at a more accurate assessment of the classification. To this end, the invention further provides means and methods to further classify individuals that suffer from arthritis or that are suspected/at risk of having arthritis. In a preferred embodiment, a method of the invention is combined with a test for Anti-Citrullinated Protein Antibodies (ACPA).

The ACPA test has been used for quite some time (reviewed, among others, in Venrooij et al. “Anticitrullinated protein/peptide antibody and its role in the diagnosis and prognosis of early rheumatoid arthritis,” The Netherlands Journal of Medicine, 2002, 60:383-388; and L. Klareskog, J. Ronnelid, K. Lundberg, L. Padyukov, and L. Alfredsson, “Immunity to citrullinated proteins in rheumatoid arthritis,” Annu. Rev. Immunol. 2008, 26:651-75.

Many different citrullinated proteins or peptides can be used in tests to detect anti-citrullinated protein antibodies (ACPA). Citrullinated filaggrin has been used to detect the so-called anti-filaggrin antibodies (AFA). In first attempts to find suitable substrates for RA auto-antibodies, a number of linear peptides containing one citrulline residue were developed. These citrullinated peptides were specifically recognized by the RA auto-antibodies and, more important, their arginine-containing counterparts were not. However, most peptides reacted with only 30 to 45% of the RA sera, although more than 75% of RA sera reacted with at least one of a total of nine peptides tested (Schellekens et al. (1998), J. Clin. Invest. 101:271-281). Although linear peptides may be used, it has been found that cyclic peptides render the tests more sensitive. Tests that include cyclic citrullinated protein/peptide are typically referred to as CCP tests. The CCP1 test was already sensitive, but it has been found that a novel selection of cyclic citrullinated protein/peptides with improved immune recognition properties has increased the sensitivity of the CCP test to at least 80%. This latter test is typically referred to as the CCP2 test. Thus, in one embodiment hereof, the method for detecting ACPA comprises detecting anti-cyclic citrullinated protein/peptide in the sample. Preferably, the method for detecting ACPA is a CCP2 test as described in G. J. Pruijn, A. Wiik, and W. J. van Venrooij, “The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis, Arthritis Res. Ther. 2010, 12(1):203, Epub. 2010 Feb. 15; and F. A. van Gaalen, H. Visser, and T. W. Huizinga, “Review and a comparison of the diagnostic accuracy and prognostic value of the first and second anti-cyclic citrullinated peptides (CCP1 and CCP2) autoantibody tests for rheumatoid arthritis,” Ann. Rheum. Dis., October 2005, 64(10):1510-2.

As is the case for ACPA, also for an anti-CarP antibody in principle, many different carbamylated proteins or peptides can be used in tests to detect anti-carbamylated protein antibodies. In a preferred embodiment, the carbamylated protein or peptide is a cyclic peptide. A good source of carbamylated proteins is carbamylated fetal calf serum. Serum proteins from other species are, however, also suitable. Following optimization, human proteins might also be used. For reasons of inevitable background, human serum cannot be used in carbamylated form without extensive depletion of Ig. In a preferred embodiment, the carbamylated protein is fibrinogen, preferably human fibrinogen.

A peptide for use in a method hereof, be it for the detection of ACPA in general or of anti-CarP antibodies or of anti-Ca-Fib antibodies, is typically a peptide of between 6 and 50 amino acids. Preferably, the peptide is a peptide of between 12 and 30 amino acids, more preferably of between 18 and 22 amino acids, most preferably of about 21 amino acids. The mentioned ranges include the number mentioned, i.e., a range of between 12 and 30 amino acids includes peptides of 12 and 30 amino acids, respectively. The peptide may or may not be a cyclic peptide depending on the sensitivity and/or specificity than the comparable linear peptide. Circular peptides can be generated in any molecular composition as to generate the cyclic nature. Any method may be used to couple peptides and or proteins in carbamylated, citrullinated or native form to plates and/or beads, being either direct coating or using biotin-streptavidin or any other available coating method.

In a preferred embodiment, such peptide for use in a method of the invention is a peptide derived from human fibrinogen. The peptide preferably comprises a contiguous amino acid of between 12 and 30 amino acids, more preferably of between 18 and 22 amino acids, most preferably of about 21 amino acids present in the amino acid sequence of any one of fibrinogen alpha (FIG. 14), fibrinogen beta (FIG. 15) or fibrinogen gamma (FIG. 16). In a more preferred embodiment, the peptide is any one of the peptides depicted in Table I, Table II, or Table III.

Further provided is, as indicated hereinabove, a method for classifying an individual that is suffering from, or at risk of suffering from, a form of arthritis, the method comprising determining whether a sample comprising a body fluid of the individual comprises an anti-CarP antibody, preferably an anti-Ca-Fib antibody, and determining whether the sample comprises Anti-Citrullinated Protein Antibodies (ACPA) and classifying the individual on the basis of the detection of the anti-CarP antibody and/or the ACPA.

The anti-CarP antibody, the anti-Ca-Fib antibody, and ACPA can be of any immunoglobulin isotype. The art typically focuses on one or more of the IgG subclasses. In the present invention, it has been found that the level of an anti-CarP antibody, anti-Ca-Fib antibody and/or ACPA of both the Ig-subtype A (IgA) and the Ig-subtype G (IgG) in a sample comprising body fluid of the individual is predictive for clinical outcome measures such as joint destruction. It has also been found that whereas a sample can be negative for anti-CarP or anti-Ca-Fib IgG, it can be positive for anti-CarP or anti-Ca-Fib IgA and vice versa. Thus, in a preferred embodiment of a method hereof, the method comprises determining whether a sample comprising a body fluid of the individual comprises an anti-CarP and/or anti-Ca-Fib antibody of Ig-subtype IgA or an anti-CarP and/or anti-Ca-Fib antibody of Ig-subtype IgG, or both, and wherein detection of the IgA and/or IgG anti-CarP and/or anti-Ca-Fib antibody indicates that the individual is suffering from, or at risk of suffering from, arthritis. In a preferred embodiment, the method further comprises determining whether a sample comprising a body fluid of the individual comprises ACPA. As mentioned hereinabove, a method of the invention is particularly useful in subdividing the heterogeneous group of ACPA-negative individuals. Using a method hereof, this ACPA-negative group can be divided into a group that is anti-CarP and/or anti-Ca-Fib antibody positive and that can be classified as suffering from, or at risk of suffering from, arthritis, and a group that is anti-CarP and/or anti-Ca-Fib antibody negative that can be classified as not suffering from arthritis or not at risk of suffering from arthritis. This determination can also be done on an individual basis. Thus, a method hereof may further comprise determining whether a sample comprising a body fluid of the individual comprises Anti-Citrullinated Protein Antibodies (ACPA) and wherein the level of the ACPA in the sample is below the detection limit and or cut-off of positivity.

A sample of an individual tested for the presence of an anti-CarP antibody, preferably of isotype IgA or of isotype IgG, or both, and ACPA can be classified on the basis of the result as an:

-   -   a) an anti-CarP and/or anti-Ca-Fib antibody positive,         ACPA-negative sample;     -   b) an anti-CarP and/or anti-Ca-Fib antibody positive,         ACPA-positive sample;     -   c) an anti-CarP and/or anti-Ca-Fib antibody negative,         ACPA-positive; and     -   d) an anti-CarP and/or anti-Ca-Fib antibody negative,         ACPA-negative sample.

Results a), b) and c) classify the sample as a sample of an individual that is at high risk to be currently suffering from, or at risk of developing, arthritis. Result d) classifies the sample as a sample of an individual that has a low risk to be currently suffering from or developing arthritis. In the event that the individual presented with undifferentiated arthritis, then classifying the sample in group d) indicates that the individual has a high chance of spontaneous remission. Such an individual is not expected to benefit on the long term from an anti-arthritis treatment. The invention further provides a method for typing a sample comprising a body fluid of an individual suffering from undifferentiated arthritis, the method comprising determining whether the sample comprises an anti-CarP antibody, preferably an anti-Ca-Fib antibody, and typing the sample as derived from an individual that has a higher than average chance of spontaneous remission of the arthritis when the sample was determined to be negative for the anti-CarP antibody, preferably the anti-Ca-Fib antibody, and typing the sample as derived from an individual that has a higher than average chance of having or progressing to RA when the sample was determined to be positive for the anti-CarP antibody, preferably the anti-Ca-Fib antibody. The higher than average chance is typically arrived at by comparison of the chance with the average chance arrived at for a number of unselected individuals presenting with undifferentiated arthritis.

In another aspect, provided is a method for estimating the severity of arthritis for an individual that is suffering from a form of arthritis and has an increased risk of developing a more severe form of arthritis, the method comprising determining whether a sample comprising a body fluid of the selected individual comprises anti-homocitrulline-Containing Protein antibodies (an anti-CarP antibody), preferably anti-homocitrulline-Containing Fibrinogen antibodies (an anti-Ca-Fib antibody), and estimating the severity of the arthritis based on the detection of the anti-CarP antibodies, preferably, the anti-Ca-Fib antibody in the sample.

In yet another aspect, provided is a method for providing a prognosis for the development of arthritis to an individual suffering from arthritis, the method comprising determining whether a sample comprising a body fluid of the individual comprises an anti-CarP antibody, preferably an anti-Ca-Fib antibody, and estimating the future severity of arthritis based on the detection of the anti-CarP antibody, preferably an anti-Ca-Fib antibody, in the sample. The method is preferably combined with a method for determining ACPA is the sample and the combined result of the anti-CarP and/or anti-Ca-Fib antibody test and the ACPA test is used to estimate the future severity of arthritis for the individual.

The sample to be tested for the presence of an anti-CarP antibody, preferably an anti-Ca-Fib antibody, and/or ACPA can, in principle, be any type of sample as long as it contains body fluid of the individual. Typically, however, the sample is a sample of body fluid. In a preferred embodiment, the sample comprising body fluid is a serum sample or a synovial fluid sample.

Arthritis is, as mentioned hereinabove, a complex disease and many different forms of arthritis have presently been identified. The arthritis that the individual is suffering from or at risk of suffering from is preferably an arthritis selected from rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, reactive arthritis, gout, pseudogout, autoimmune arthritis, systemic lupus erythematosus, polymyositis, fibromyalgia, Lyme disease, undifferentiated arthritis, non-rheumatoid arthritis or spondyloarthropathy. More preferably, the arthritis that the individual is suffering from or at risk of suffering from is an arthritis selected from rheumatoid arthritis, psoriatic arthritis, osteoarthritis, polymyalgia rheumatica, ankylosing spondylitis, reactive arthritis, gout, pseudogout, autoimmune arthritis, systemic lupus erythematosus, polymyositis, fibromyalgia, Lyme disease, undifferentiated arthritis, non-rheumatoid arthritis or spondyloarthropathy. Preferably, the arthritis is selected from rheumatoid arthritis, juvenile arthritis, more preferably juvenile idiopathic arthritis, or undifferentiated arthritis. More preferably, the arthritis is selected from rheumatoid arthritis or undifferentiated arthritis.

Further provided is a method for typing the arthritis of an individual suffering from a form of arthritis, the method comprising determining whether a sample comprising a body fluid of the individual comprises an anti-Carbamylated Protein (anti-CarP) antibody, preferably an anti-Carbamylated Fibrinogen (anti-Ca-Fib) antibody. The arthritis can be typed on the basis of the detected presence or absence of the anti-CarP and/or anti-Ca-Fib antibody. A sample in which the anti-CarP antibody, preferably the anti-Ca-Fib antibody, is detected is more likely to be derived from an individual with rheumatoid arthritis. Thus, this method can be used alone, or in combination with another test for RA to assess the likelihood that the individual is suffering from RA. In a preferred embodiment, the further test comprises a test for the presence of ACPA, preferably a CCP2 test and or a test for Rheumatoid Factor.

Shown herein is that anti-CarP antibodies can be detected in asymptomatic, healthy individuals before RA development. Thus, detection of anti-CarP antibodies are also useful in the healthy population for the early identification of patients at risk to develop RA or persistent arthritis later in life. In addition, the detection of anti-CarP antibodies is useful to identify persons that would benefit from early treatment, preferably before they fulfill the current classification for RA.

In a further aspect, provided is a method for determining whether an individual is at risk of developing or suffering from a form of arthritis, and wherein the individual was not known to suffer, or preferably not suffering from, arthritis at the time that the sample comprising body fluid was collected, the method comprising determining whether a sample comprising a body fluid of the individual comprises an Anti-Carbamylated Protein (anti-CarP) antibody. In a preferred embodiment, a method hereof is provided wherein the anti-CarP antibody is an Anti-Carbamylated Fibrinogen (anti-Ca-Fib) antibody. A sample in which the anti-CarP antibody, preferably the anti-Ca-Fib antibody, is detected is likely to be derived from an individual that is at risk of suffering from or developing arthritis, in particular RA, in the near future, particularly within five years from the date of sample collection, particularly within three years and more particularly within one and a half years from the date of sample collection. This method can be used alone, or in combination with, another test for RA to assess the risk. In a preferred embodiment, the further test comprises a test for the presence of ACPA, preferably a CCP2 test and/or a test for Rheumatoid Factor.

In another aspect, provided is a kit for the detection of anti-CarP antibodies, preferably anti-Ca-Fib antibodies, in a body fluid of an individual, the kit comprising a carbamylated protein or peptide. Preferably, the carbamylated protein or peptide is carbamylated fibrinogen or a fibrinogen-derived peptide. In a preferred embodiment, the protein or peptide is a carbamylated protein or peptide as indicated hereinabove. In particular, the kit comprises at least one peptide comprising a contiguous amino acid of between 12 and 30 amino acids, more preferably of between 18 and 22 amino acids, most preferably of about 21 amino acids, present in the amino acid sequence of any one of fibrinogen alpha (FIG. 14), fibrinogen beta (FIG. 15) or fibrinogen gamma (FIG. 16). In a more preferred embodiment, the kit comprises at least one of the peptides depicted in Table I, Table II, or Table III.

As also mentioned hereinabove, in a preferred embodiment, the kit further comprises an anti-human IgG antibody and/or an anti-human IgA antibody. Preferably, the anti-human Ig antibody comprises a label that can be detected. Non-limiting examples of such labels are a direct labeling with HRP or AP. Alternatively, the kit preferably further comprises another antibody, which antibody is specific for the anti-human Ig antibody used. In this embodiment, another antibody comprises a label that can be detected, such as biotin or DIG. This nesting can, of course, be continued with label being present on the last and/or one or more (earlier) antibodies. In a preferred embodiment, the anti-human Ig antibody comprises an anti-human IgA antibody. In a more preferred embodiment, the kit comprises an anti-human IgA antibody and an anti-human IgG antibody. The anti-human Ig antibody, as indicated hereinabove, is typically a full antibody; however, a fragment that contains the antigen binding site is also within the use of the term “antibody.” Similarly, there are presently a great variety of different binding proteins or peptides available that can be tailored to specifically bind to human Ig. Such human Ig-specific binding proteins or peptides are equivalents of an anti-human Ig antibody as defined herein. Similarly, the one or more further antibodies in a nesting setting can be replaced with binding proteins and/or peptides that specifically bind the earlier antibody and/or binding protein/peptide in the nesting tree.

In a particularly preferred embodiment, the kit further comprises a citrullinated protein or peptide. In a preferred embodiment, the protein or peptide is a citrullinated protein or peptide as indicated hereinabove. As also mentioned hereinabove, the citrullinated protein or peptide is preferably a protein or cyclic peptide. Preferably, the kit comprises a cyclic citrullinated peptide of a CCP1 or CCP2 test. Preferably, the kit comprises all cyclic peptides of a CCP test, preferably a CCP2 test.

A method hereof is, as mentioned hereinabove, preferably combined with another arthritis classifier test. Thus, in a preferred embodiment of a method hereof, the method further comprises determining a further factor as an arthritis classifier for the individual. Preferably, the further factor comprises determining ACPA, rheumatoid factor, C-reactive protein, and/or erythrocyte sedimentation rate.

Carbamylation is defined herein as the process of providing a protein or peptide with a modification that generates a homocitrulline residue. In this document where reference is made to a “carbamylated protein or peptide or collection thereof,” reference is made to the protein or peptide having a homocitrulline modification, or a collection of proteins or peptide having homocitrulline modifications.

Further provided is an anti-CarP antibody, preferably an anti-Ca-Fib antibody, and ACPA for use in determining whether an individual is suffering from, or at risk of suffering from, a form of arthritis. The invention further provides an anti-CarP antibody, preferably an anti-Ca-Fib antibody, and ACPA for use in the classification of arthritis, the determining and/or classification being a determining or classification as indicated hereinabove.

Further provided is a method for determining whether an individual that is suffering from a form of arthritis has an increased risk of developing a more severe form of arthritis, the method comprising selecting an individual that is suffering from arthritis but that does not have the most severe form of arthritis and determining whether a sample comprising a body fluid of the selected individual comprises an anti-Carbamylated Protein Antibody (an anti-CarP antibody), wherein detection of the anti-CarP indicates that the individual has increased risk of developing a more severe form of arthritis. In a preferred embodiment, a method according to the invention is provided, wherein the anti-CarP antibody is an anti-Carbamylated Fibrinogen Antibody (an anti-Ca-Fib antibody).

In this document where reference is made to “detection, determination or other assessment” of an anti-CarP antibody, an anti-Ca-Fib antibody or an ACPA, the reference includes that more than one anti-Carp antibody, anti-Ca-Fib antibody and/or more than one ACPA is detected, determined or otherwise assessed. Anti-CarP or anti-Ca-Fib antibody can recognize a homocitrulline modification per se or, more typically, recognize the modification in the context of one or more of the amino acids of the protein or peptide in the immediate vicinity of the homocitrulline modification. A method or kit of the invention is more accurate when anti-CarP antibodies and/or anti-Ca-Fib antibodies of more than one specificity is detected, determined or otherwise assessed. A method of the invention thus preferably comprises determining two or more, and preferably three or more, more preferably five or more, most preferably at least seven anti-CarP and/or anti-Ca-Fib antibodies in the sample. Similarly, a kit of the invention preferably comprises two or more, and preferably three or more, more preferably five or more, most preferably at least seven carbamylated proteins and/or peptides, preferably fibrinogen and/or fibrinogen-derived peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1C: Development of a novel and specific assay for the detection of anti-CarP antibodies. (FIG. 1A) Dose response curves of the anti-CarP antibody-positive standard on carbamylated FCS and native FCS in ELISA. (FIG. 1 B) Inhibition studies where anti-CarP antibody binding to ELISA plates coated with Ca-FCS was inhibited using pre-incubations with fluid-phase inhibitors as indicated. Only Ca-FCS inhibited the binding of anti-CarP antibodies. (FIG. 1C) Coomassie staining showing equal loading of Ca-FCS and FCS, and Western blot showing a positive staining of Ca-FCS-loaded lanes and not FCS-loaded lanes by a serum sample of an anti-CarP-positive sample and not by a negative sample.

FIG. 2: Anti-CarP IgG and IgA antibodies are present in RA sera. ELISA was performed for the detection of anti-CarP IgG and IgA in sets of sera of healthy controls and RA patients. A cut-off was established using the mean plus two times the standard deviation of the healthy controls. Shown is the titer expressed as arbitrary units per ml following calculation based on the standard curve. Below the graph, both the number of samples tested and the percentage positivity is indicated.

FIG. 3: Anti-CarP antibodies and ACPA are two separate autoantibody systems. Pie charts showing the percentages of RA patients positive and negative for ACPA and/or anti-CarP antibodies.

FIG. 4: Anti-CarP IgA antibodies are associated with the conversion of undifferentiated arthritis (UA) to RA. Sera of patients that presented with UA at baseline were measured for anti-CarP IgG and IgA antibodies and analyzed for their conversion toward RA at the one-year follow-up. Data shown are also split up on the basis of ACPA positivity. IgA anti-CarP antibodies associate strongly with development of RA from a UA population.

FIG. 5: Anti-CarP IgA antibodies are associated with more severe radiological progression in RA. The extent and rate of joint destruction were analyzed in the RA patients split up on the basis of positivity for ACPA and anti-CarP antibodies. Positivity for anti-CarP IgA antibodies is associated with a more severe radiological damage in both ACPA-negative and ACPA-positive RA patients.

FIGS. 6A and 6B: Illustration of citrullination and carbamylation. Citrullination and carbamylation occur on different amino acids via different mechanisms, but yield similar end-products.

FIG. 7: Antibodies against carbamylated proteins are present in sera of RA patients. The reactivity of IgG (Panels A and B) and IgA (Panels D and E) from sera of healthy controls (NHS) or RA patients (RA) to wells coated with non-modified FCS (FCS) or carbamylated FCS (Ca-FCS) is depicted. Data expressed as absorbance at 415 nm. In Panels C and F, absorbance units of FCS were subtracted from the absorbance units of Ca-FCS, representing the specific anti-carbamylated protein response.

FIG. 8: Anti-CarP antibodies and ACPA are two separate autoantibody systems. Panel A depicts IgG reactivity of 76 sera from RA patients toward several forms of a Fib peptide. Panels B and C depict binding to Ca-FCS or Ci-FCS was inhibited using pre-incubations with fluid-phase inhibitors. Panel D shows that FCS, Ca-FCS and Ci-FCS were separated by SDS-page gels and blotted. The presence of antibodies reactive to proteins on the blots was analyzed by incubating these blots with either anti-CarP-positive ACPA-negative and anti-CarP-negative ACPA-positive sera.

FIG. 9: Anti-CarP antibodies bind to Ca-Fib via variable domains. Panel A: IgG reactivity against Fib, Ci-Fib and Ca-Fib of 54 healthy controls and 214 RA patients was analyzed by ELISA. Panel B: Specificity of anti-Ca-Fib reactivity was confirmed using inhibition studies. One sample is shown, where data are expressed relative to inhibition with PBS. Panel C: The molecular nature of purified IgG and F(ab′)2 was confirmed by Coomassie-stained SDS page gel. Panel D: F(ab′)2 fragments were generated from purified IgG of two anti-CarP-positive patients and two negative controls. Only F(ab′)2 from patients reacted with Ci-Fib and Ca-Fib. Panel E: Inhibition experiments also confirm that F(ab′)2 are not necessarily cross-reactive between Ci-Fib and Ca-Fib.

FIG. 10: Anti-CarP IgG and IgA antibodies are present in RA sera. Panels A and B: Dose response curves of the anti-CarP antibody-positive standard (IgG and IgA) on Ca-FCS and FCS in ELISA. Panels C and D: ELISA was performed for the detection of anti-CarP IgG and IgA in sera of healthy controls (NHS) and RA patients. A cut-off was established using the mean plus two times the standard deviation of the healthy controls as described in the methods. Reactivity is depicted as arbitrary units per mL. The number of samples tested and the % positivity is indicated below the graph. Panels E and F: Pie charts showing the % of RA patients positive and negative for anti-CCP2 and/or anti-CarP antibodies. Panels G and H: Pie charts showing the % of anti-CarP IgG- or IgA-positive patients negative for anti-CCP2.

FIG. 11: Anti-CarP IgG antibodies are associated with a more severe radiological progression in ACPA-negative RA. The extent and rate of joint destruction were analyzed in all RA patients included, or analyzed separately for ACPA-negative or ACPA-positive subgroups (FIG. 13). The severity of joint destruction is depicted as median Sharp/van der Heijde score (SHS) on the Y-axis and the follow-up years on the X-axis. The patient number is listed for each time point below the X-axis. Radiological progression for the anti-CCP2-negative RA patients is shown. The P-value is derived from the analysis model as described in the methods section.

FIGS. 12A through 12C: Anti-CarP IgG antibodies are associated with the conversion of pre-disease to RA. FIG. 12A: Sera of patients that presented with Undifferentiated Arthritis (UA) at baseline were measured for anti-CarP IgG antibodies and analyzed for their conversion toward RA at 1 year follow-up. Data shown are also split up on the basis of ACPA positivity. Anti-CarP antibodies associate strongly with development of RA from a UA population. FIG. 12B: Also in sera of patients that presented with arthralgia at baseline, anti-CarP antibodies are predictive for development of RA. FIG. 12C: Sera of healthy persons that do not develop RA or that do develop RA later in life are compared for anti-CarP positivity. The presence of anti-CarP is associated with future development of RA.

FIG. 13: Anti-CarP IgA antibodies are associated with more severe radiological progression in RA. The extent and rate of joint destruction were analyzed in the RA patients split up on the basis of positivity for ACPA and anti-CarP antibodies. Positivity for anti-CarP IgG antibodies is associated with a more severe radiological damage in ACPA-negative RA patients.

FIG. 14: Amino Acid Sequence of Fibrinogen alpha (SEQ ID NO:1).

FIG. 15: Amino Acid Sequence of Fibrinogen beta (SEQ ID NO:2).

FIG. 16: Amino Acid Sequence of Fibrinogen gamma (SEQ ID NO:3).

FIG. 17: Anti-CarP antibodies are present in sera of patients suffering from juvenile arthritis. ELISA was performed for the detection of anti-CarP IgG in sera of healthy children (Ctr) and in sera of patients suffering from juvenile arthritis. A cut-off was established using the mean plus two times the standard deviation of the healthy controls as described in the methods. Reactivity is depicted as arbitrary units per mL. The number of samples tested and the % positivity is indicated below the graph.

DETAILED DESCRIPTION EXAMPLES Example 1

Materials and Methods

Generation of Antigens

As a source of antigens, we have used fetal calf serum (FCS) (Bodinco, batch No. 212-192909). This was carbamylated, citrullinated or employed as an unmodified source.

Carbamylated FCS (Ca-FCS) was generated by diluting FCS in bidest to 4 mg/ml. Potassium cyanate (sigma, Cat. No. 215074) was added at 80 mg/ml. Following incubation at 37° C. for 12 hours, the sample was extensively dialyzed against bidest.

As a control, citrullinated FCS (Ci FCS). For this purpose, 50 μl FCS (24 mg/me with 24 μl 0.5 M Tris-HCl pH 7.6+15 μl 0.125 M CaCl₂+31 μl PAD4 (Sigma P1584) was incubated for 24 hours at 37° C.

Detection of Anti-CarP Antibodies by ELISA

Non-modified FCS and Ca-FCS were coated at 10 μg/ml (diluted in pH 9.6 0.1 M carbonate-bicarbonate buffer) 50 μl on Nunc immunoplates (Thermo Scientific, Cat. No. 430341), overnight at 4° C. Following washing for four times in phosphate-buffered saline (PBS) containing 0.05% TWEEN® (Sigma, Cat. No. 27,434-8) (PT), the plates were blocked by incubating 100 μl PBS/1% bovine serum albumin (BSA) (Sigma, Cat. No. A2153) for 1 hour at 37° C. Following additional washing, the sera were incubated in 50 μl at a 1/50 (in PBS/0.05% TWEEN®/1% BSA buffer (PTB)) to both FCS- and Ca-FCS-coated wells and incubated at 37° C. for 1 hour. Serial dilutions of a standard serum (diluted in PTB) were incubated on Ca-FCS-coated wells. Following washing, bound human IgG or IgA was detected by incubating the wells with 50 μl 1/5000 diluted (in PTB) rabbit anti-human IgG antibody (Dako, Cat. No. A0423) or 1/1000 diluted (in PTB) rabbit anti-human IgA antibody (Dako, Cat. No. A0262) incubated at 37° C. for 1 hour. Following washing, wells were incubated at 37° C. for 1 hour with 50 μl 1/2000 diluted (in PTB) goat anti-rabbit IgG HRP-labeled antibody (Dako, Cat. No. PO448). Following the last washings, HRP enzyme activity was visualized by incubating 50 μl 2,2′Azino-bis-(3-ethylbenzo-thiazole-6-sulfonic acid) diammonium salt (ABTS) and H₂O₂, measuring absorbance at 415 nm on a standard ELISA reader.

Detection of Anti-CarP Antibodies by Western Blot

Both FCS and Ca-FCS were loaded onto regular 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto Hybond-C Extra membranes (Amersham, Diegem, Belgium). Blots were then incubated in blocking buffer (3% ELK Milk/PBS/0.05% TWEEN®) 1 hour at RT, following washing with PBS/0.05% TWEEN®. The blots were subsequently incubated with 5 ml serum 1:500 diluted in blocking buffer for 1 hour at RT. After three washes with PBS/0.05% TWEEN®, blots were incubated with 3 ml horseradish peroxidase conjugated rabbit anti-human IgG (DAKO, Heverlee, Belgium) 1:50,000 diluted in blocking buffer for 1 hour at RT. Next, blots were washed and bound antibodies were visualized using enhanced chemiluminescence (ECL; Amersham). Equal protein loading was verified using Coomassie Brilliant Blue (Bio-Rad, Veenendaal, The Netherlands).

Sera and Synovial Fluids

The sera analyzed were from patients participating in the Leiden Early Arthritis Clinic (EAC) cohort. The Leiden EAC is an inception cohort of patients with recent-onset arthritis (symptoms duration <2 years) that was started at the Department of Rheumatology of the Leiden University Medical Center in 1993.⁽¹⁵⁾ All RA patients fulfilled the American College of Rheumatology (formerly the American Rheumatism Association) 1987 revised criteria for the classification of RA⁽¹⁶⁾ within one year of follow-up (EAC cohort). A total of 1007 patients were analyzed, of which 582 were diagnosed as RA and 425 as UA, of which 151 developed RA on follow-up. These patient samples were compared to 280 healthy control samples also derived from the Leiden area. An additional set of paired serum/synovial fluid of RA patients was analyzed. The protocols were approved by the relevant local ethics committee and all participants provided informed consent.

ELISA for the Detection of ACPA

Total IgG anti-CCP2, as a measure of ACPA, was measured in sera collected at baseline by enzyme-linked immunosorbent assay (ELISA) (Immunoscan RA Mark 2; Eurodiagnostica, Arnhem, The Netherlands). Samples with a value above 25 units/ml were considered positive according to the manufacturer's instructions. Individuals with antibodies against CCP2 were considered ACPA-positive.

ELISA for the Detection of Anti-CaFib Antibodies

Non-modified Fib and Ca-Fib were coated at 20 μg/ml in 50 μl (diluted in pH 9.0 PBS) on Nunc Maxisorp plates ON. Following washing in PBS TWEEN®, the plates were blocked by incubating 200 μl pH 9.0 PBS/2% BSA for 2 hours at 4° C. Following additional washing, the wells were incubated with 50 μl serum at a 1/50 dilution in RIA buffer (10 mM Tris pH 7.6; 350 mM NaCl; 1% TritonX; 0.5% Na-deoxycholate; 0.1% SDS) (Sigma) on ice for 3 hours. All subsequent incubations are performed in RIA buffer. As a standard, serial dilutions of a pool of positive sera were used. Human IgG was detected using HRP-labeled rabbit anti human IgG antibody (DAKO) incubated on ice for 2 hours. Following the last washings, HRP enzyme activity was visualized using ABTS. We transformed the absorbance on Fib and Ca-Fib to aU/mL. We established the cut-off for a positive response as the mean plus 2×the standard deviation of the specific anti-CarP reactivity of the healthy controls.

We analyzed 67 sera of healthy children and 110 sera of patients suffering from juvenile arthritis.

Statistics

Data were analyzed using the Statistical Package for the Social Sciences (SPSS) 17.0 using logistic regression. P-values below 0.05 were considered to be statistically significant.

Example 2

Materials and Methods

Patient and Control Sera

The sera analyzed were from patients participating in the Leiden Early Arthritis Clinic (EAC) cohort. The Leiden EAC is an inception cohort of patients with recent-onset arthritis (symptoms duration <2 years) that was started at the Department of Rheumatology of the Leiden University Medical Center in 1993.⁽³⁹⁾ All RA patients fulfilled the American College of Rheumatology (formerly the American Rheumatism Association) 1987 revised criteria for the classification of RA⁽⁴⁰⁾ within 1 year of follow-up. A total of 571 RA patients were involved in the analyses. Patient samples were compared to 305 healthy control samples also living in the Leiden area. The protocols were approved by the local ethics committee and informed consent was obtained.

Detection of Anti-CarP Antibodies by ELISA

In brief, Non-modified FCS and modified-FCS were coated on NUNC MAXISORP™ plates (Thermo Scientific) over night. Following washings and blocking, the wells were incubated with serum. Bound human IgG or IgA was detected using rabbit anti-human IgG or IgA antibodies (DAKO), followed by HRP-labeled goat anti-rabbit IgG 4bb ABTS.⁽⁴¹⁾ A more detailed description of the protein modifications and ELISA assays based on FCS and Fib, including F(ab)2, is available online (SI-materials and Methods). The cut-off for a positive response was established as the mean plus 2×the standard deviation of the specific anti-CarP reactivity of the healthy controls. The methods for the detection of ACPA and Western blotting are available online (SI-materials and Methods).

ELISA for Fib Peptides

Streptavidin (Invitrogen) was coated at 2 μg/ml in 100 μl on Nunc plates at 4° C. ON. After washing, Fib peptides containing either an arginine, citrulline, homocitrulline or a lysine (FIG. 8, Panel A)⁽⁴²⁾ were incubated at 10 μg/ml in 100 μl PTB for 1 hour at RT. Next, the reactivity of antibodies reactive to these antigens was detected as described above.

Inhibition Studies

To determine whether anti-CarP antibodies and ACPA are cross-reactive antibodies, we performed inhibition studies in which autoantibody-positive serum samples, positive for both ACPA and anti-CarP antibodies, were pre-incubated with increasing concentrations of either non-modified FCS, Ca-FCS, Ci-FCS or the citrulline- or arginine-containing form of the CCP1 peptide.⁽⁴³⁾ Following pre-incubation at room temperature (RT), the samples were tested for reactivity against Ca-FCS and Ci-FCS as described above. Serum and F(ab′)2 samples positive for both Ci-Fib and Ca-Fib were pre-incubated with Fib, Ci-Fib and Ca-Fib at 4° C. ON and subsequently analyzed on the Fib ELISA (SI-materials and Methods).

Radiological Progression

In the EAC cohort, radiographs of the hands and feet, which had been obtained in a longitudinal fashion, were scored according to the Sharp/van der Heijde method.⁽⁴⁴⁾ Scoring and analysis have been described in detail before.⁽²¹⁾ Data are analyzed directly, or using repeated measurement analysis, as to optimally make use of the longitudinal data obtained for each patient.⁽²¹⁾ More detailed information is available online (SI-materials and Methods).

Generation of Antigens

Because it was not known whether antibodies against carbamylated proteins would be present in sera of RA patients, or which proteins they would recognize, a study of a diverse set of carbamylated proteins was commenced in order to maximize the chances of detecting as many of the anti-CarP reactivities as possible. For this purpose, fetal calf serum (FCS) (Bodinco) that was carbamylated, citrullinated or left untreated was used. For generating carbamylated FCS (Ca-FCS), FCS was diluted in H₂O to 4 mg/ml and potassium cyanate (Sigma) was added to a concentration of 1 M. Following incubation at 37° C. for 12 hours, the sample was extensively dialyzed against H₂O. Carbamylated fibrinogen (Ca-Fib) was generated by incubating 5 mg/ml fibrinogen (Fib) with 0.5 M potassium cyanate at 4° C. for three days, followed by extensive dialysis against PBS. Citrullinated FCS (Ci-FCS) and citrullinated fibrinogen (Ci-Fib) was generated by incubation of 10 mg FCS or Fib in a volume of 1 mL containing 0.1 M Tris-HCl pH 7.6, 0.015 M CaCl₂ and 40 U PAD4 (Sigma) for 24 hours at 37° C. The presence of citrulline and homocitrulline residues was confirmed using mass-spectrometry analysis. For Fib, extensive citrullination and complete carbamylation was observed in the protein segments analyzed.

Detection of Anti-CarP Antibodies by ELISA

Non-modified FCS and modified-FCS were coated at 10 μg/ml in 50 μl (diluted in pH 9.6 0.1 M carbonate-bicarbonate buffer) (CB) on NUNC MAXISORP™ plates (Thermo Scientific) overnight (ON). Following washing in PBS containing 0.05% TWEEN.RTM. (Sigma) (PT), the plates were blocked by incubating 100 μl PBS/1% bovine serum albumin (BSA) (Sigma) for 6 hours at 4° C. Following additional washing, the wells were incubated with 50 μl serum at a 1/50 dilution in PBS/0.05% TWEEN.RTM./1% BSA buffer (PTB) on ice overnight. All subsequent incubations are performed in PTB. As a standard, serial dilutions of a pool of positive sera were used. Human IgG or IgA was detected using rabbit anti-human IgG antibody (DAKO) or rabbit anti-human IgA antibody (Dako) incubated on ice for 3.5 hours. Following washing, wells were incubated on ice for 3.5 hours with HRP-labeled goat anti-rabbit IgG antibody (Dako). Following the last washings, HRP enzyme activity was visualized using ABTS as described before. ⁽²²⁾ Sera of healthy subjects (n=305) were used as controls. The absorbance on both Ca-FCS and FCS was transformed to aU/mL and subtracted the background signal (aU/mL) of FCS from the signal (aU/mL) of Ca-FCS as to analyze the specific anti-CarP reactivity (FIG. 7). The cut-off was established for a positive response as the mean plus 2×the standard deviation of the specific anti-CarP reactivity of the healthy controls.

ELISA for Fibrinogen

Non-modified Fib Ci-Fib and Ca-Fib were coated at 20 μg/ml in 50 μl (diluted in pH 9.0 PBS) on NUNC MAXISORP™ plates ON. Following washing in PT, the plates were blocked by incubating 200 μl pH 9.0 PBS/2% BSA for 2 hours at 4° C. Following additional washing, the wells were incubated with 50 μl serum at a 1/50 dilution in RIA buffer (10 mM Tris pH 7.6; 350 mM NaCl; 1% TritonX; 0.5% Na-deoxycholate; 0.1% SDS) (Sigma) on ice for 3 hours. All subsequent incubations are performed in RIA buffer. As a standard, serial dilutions of a pool of positive sera were used. Human IgG was detected using HRP-labeled rabbit anti-human IgG antibody (DAKO) incubated on ice for 2 hours. Following the last washings, HRP enzyme activity was visualized using ABTS. Sera of 214 RA patients and 54 healthy subjects as controls was analyzed. The absorbance on Fib Ci-Fib and Ca-Fib was transformed to aU/mL. The cut-off was established for a positive response as the mean plus 2×the standard deviation of the specific anti-CarP reactivity of the healthy controls. These assays were repeated three times showing the same data.

F(ab′)2 Preparation

Total IgG from two anti-CarP-positive and two control sera were isolated via a HiTrap™ protein A HP column (GE Healthcare) following the protocol for the column provided by the manufacturer. F(ab′)2 fragments were generated from purified IgG samples using a F(ab′)2 Preparation Kit (Thermo Scientific) following the protocol provided by the manufacturer. The molecular nature of the intact IgG and the F(ab′)2 was verified using Coomassie-stained SDS page gels. These F(ab′)2 were used in ELISA as described above, now using either HRP-labeled rabbit anti-human IgG, IgA, IgM kappa, lamda antibody (anti-light chain) (Dako) or HRP-labeled rabbit anti-human IgG (Dako).

Detection of ACPA by ELISA

ACPA were measured by the CCP2 ELISA (Immunoscan RA Mark 2; Eurodiagnostica, Arnhem, The Netherlands). Samples with a value above 25 units/ml were considered positive according to the manufacturer's instructions. A small percentage of ACPA-positive RA patients may be outside the anti-CCP2 reactivity and, therefore, both terms will be used to explicitly indicate what has been used in our analyses.

ACPA reactivity toward Ci-FCS was detected using ELISA plates that were coated with Ci-FCS (50 μl/well 10 .mu.g/ml) diluted with CB in the NUNC MAXISORP™ plates ON at 4° C. The plates were washed in PT followed by blocking with 100 μl PBS/1% BSA solution at 37° C. for 1 hour. Following washing, sera were incubated at a 1/50 dilution in 50 μl PTB and incubated at 37° C. for 1 hour. After washing, human IgA and IgG were detected as described above.

Detection of Anti-CarP Antibodies by Western Blot

FCS, Ca-FCS and Ci-FCS were loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto Hybond-C Extra membranes (Amersham). Blots were incubated in blocking buffer (3% ELK Milk/PBS/0.05% TWEEN®) for 1 hour at RT, following washing with PT. The blots were subsequently incubated with 2.5 ml 1:500 diluted serum in blocking buffer for 1.5 hours at RT. The sera were either ACPA-positive anti-CarP negative or ACPA-negative anti-CarP positive as determined by ELISA. After three washes with PT, blots were incubated with 5 mL HRP-labeled rabbit anti-human IgG diluted in blocking buffer for 1 hour at RT. Next, blots were washed and bound antibodies were visualized using enhanced chemiluminescence (Amersham).

Statistics of Radiological Progression

Association between anti-CarP antibodies positivity and radiographic progression was analyzed using the Statistical Package for the Social Sciences (SPSS) 17.0 as described before. P-values below 0.05 were considered statistically significant. A multivariate normal regression analysis for longitudinal data was used with radiological score as response variable. This method analyses repeated measurements at once and takes advantage of the correlation between these measurements, which results in a more precise standard error. Radiological scores were log-transformed to obtain a normal distribution. The rate of joint destruction over time was tested by an interaction of time with anti-CarP. The effect of time was assumed to be linear in the interaction term. The effect of time was entered as factor in the model as well, allowing a mean response profile over time. Age, gender and inclusion period as proxy for treatment were included as correction variables in all analyses. In a separate analysis, the effect of anti-CarP antibodies was corrected for the effect of anti-CCP and RF.

Example 1

Results

Detection of Anti-CarP Antibodies

A novel ELISA was generated to detect anti-CarP antibodies from serum and synovial fluid using plates coated with, in vitro-generated, carbamylated FCS. A standard was generated from a pool of positive sera showing a dose-dependent binding of both IgG and IgA to Carbamylated FCS (Ca-FCS) and no binding to the native FCS (FIG. 1A). Since homocitrulline and citrulline are very similar residues but differ from each other by one atom, excluding the possibility that the anti-CarP antibodies were actually ACPA was desired. As a first test to exclude this, inhibition studies were performed that show that anti-CarP antibody binding to Ca-FCS can only be inhibited by Ca-FCS itself and not by citrullinated FCS (Ci-FCS), native FCS or by peptides used to detect ACPA (FIG. 1B), indicating that anti-CarP is truly a different reactivity. In addition, binding of ACPA to CCP2 plates was not inhibited by addition of Ca-FCS (data not shown).

Since these methods rely on ELISA, confirmation of those findings using Western blotting was desired. FCS and Ca-FCS was run on non-reducing gels, and following Western blotting, stained the blots using sera of patients that were positive or negative for anti-CarP antibodies as detected by ELISA. Serum of anti-CarP antibody-positive patients tested positive on lanes loaded with Ca-FCS, while no reactivity was seen in lanes loaded with FCS (FIG. 1C). Sera from anti-CarP antibody-negative patients did not show such staining. Using Coomassie staining on loaded gels, it was confirmed that the FCS-loaded lanes did contain similar protein amounts. Collectively, these data indicate that a novel assay has been generated that can specifically detect anti-CarP antibodies, both IgG and IgA.

Anti-CarP Antibodies are Present in RA

From the Leiden Early Arthritis Clinic (EAC), patients suffering from UA or RA have been used according to the 1987 inclusion criteria. In addition, healthy controls were also used from the Leiden region. The presence of anti-CarP antibodies in patients and controls was measured simultaneously. OD values were calculated to arbitrary units per mL using a standard. Healthy persons were used to calculate the cut-off for positivity as defined by the mean plus 2×the standard deviation of the healthy controls. Samples were considered to be positive when they had a titer higher than the cut-off and an absorbance that was at least 0.1 units higher on Ca-FCS as compared to FCS.⁽¹⁷⁾ Using this approach, it was established that 42% of the RA patients were positive for IgG anti-CarP antibodies, whereas 54% of sera from RA patients tested were positive for IgA anti-CarP-antibodies (FIG. 2). Analysis of paired serum/synovial fluid samples revealed that anti-CarP IgG and IgA can also be found in synovial fluid of patients that are positive for these autoantibodies in serum (data not shown).

Anti-CarP antibodies are present in serum and synovial fluid in a substantial proportion of RA patients.

Anti-CarP Antibodies are Independent from ACPA

Next, it was analyzed as to whether the anti-CarP antibodies occur independently of ACPA. To this end, the relationship between ACPA and anti-CarP antibodies in a set of 373 RA patients was analyzed. The data show that 14% and 23% of the RA patients did not display ACPA but did harbor anti-CarP IgG and IgA, respectively. Likewise, 22% and 19% of the RA patients were positive for ACPA but negative for anti-CarP IgG and IgA, respectively (FIG. 3). Zooming in on the ACPA-negative individuals, it was observed that around 50% of all ACPA-negative RA patients tested positively for anti-CarP antibodies. Thus, anti-CarP antibodies occur independently from ACPA.

Anti-CarP Antibodies are Predictive for Development of RA

Patients presenting themselves at baseline with a diagnosis of undifferentiated arthritis can go into remission, develop another form of arthritis or can develop RA. Clinically, it would be relevant to be able to discriminate between the patients in need for treatment and the patients that will remit spontaneously. Therefore, 425 patients that had UA at baseline for the presence of anti-CarP antibodies and the development of RA (n=151) were analyzed. It was observed that positivity for IgG anti-CarP antibodies did not associate with RA development according to the 1987 criteria in a statistically significant manner (p=0.11). In contrast, IgA anti-CarP antibodies were strongly associated with future development of RA in the UA group as a whole (p=0.002) (FIG. 4). This effect was especially prominent in the ACPA-negative individuals (FIG. 4).

Measuring anti-CarP antibodies in patients suffering from undifferentiated arthritis is useful to identify persons at risk to develop RA.

Anti-CarP Antibodies are Associated with More Severe Radiological Damage

Finally, analysis was performed to ascertain whether RA patients that are positive at baseline for anti-CarP antibodies would have a different clinical course of their disease. Therefore, comparisons were made as to the extent of joint damage over time. Also in this analysis, positivity for IgG anti-CarP antibodies did not associate with radiological damage in a statistically significant manner (p=0.43). However, IgA anti-CarP antibodies are strongly associated with more severe damage to the joints (p=0.002) (FIG. 5), an effect that was independent from Rheumatoid Factor (RF) or ACPA. Together, these data indicate that anti-CarP antibodies are associated with a more severe disease course, independent of the presence of RA and or ACPA.

Discussion

A novel family of autoantibodies that recognize carbamylated proteins (anti-CarP) is described. These anti-CarP antibodies can be detected in both the IgG and IgA isotypes. Both inhibition studies and cohort studies show that anti-CarP antibodies are different from ACPA. Interestingly, positivity for anti-CarP, especially IgA, has clinical implications as individuals positive for anti-CarP IgA have an increased risk to progress from UA to RA and anti-CarP IgA-positive RA patients have a worse outcome compared to anti-CarP IgA-negative RA patients.

A complex protein mix was used as an initial source of carbamylated protein antigens and, therefore, generated Ca-FCS. It was observed that antibodies exist that are specifically directed against the carbamylated form of FCS, which do not bind to native or citrullinated FCS in both ELISA and Western blot systems. These antibodies are of both the IgG and IgA isotypes, indicating that they are derived from class-switched B cells, a process that would require T cell help. Indeed, data indicate that homocitrulline directed T cells can be induced by immunization with carbamylated model antigens.⁽¹¹⁾

It is shown herein that detection of these antibodies in early arthritis can predict the future development of RA and predict a more severe disease course. Since it has been shown that early aggressive treatment is beneficial,^((18, 19)) the invention provides methods for arthritis treatment of individuals suffering from, or at risk of suffering from, arthritis, the method comprising an arthritis diagnosis of the individual wherein the diagnosis comprises a method for determining an anti-CarP antibody in a sample comprising a body fluid of the individual. Preferably, the sample was determined to contain an anti-Carp antibody. A more stringent treatment of the anti-CarP-positive individual is beneficial to the patient.

In conclusion, next to the autoantibody system that recognizes citrullinated proteins (ACPA), an autoantibody system is also present against carbamylated proteins (anti-CarP). Detection of such antibodies is useful since its presence is, independently of ACPA, associated with development of UA to RA and is associated with a more severe disease course.

Example 2

Results

Anti-CarP Antibodies and ACPA are Different Antibody Families

To detect antibodies against carbamylated proteins (anti-CarP antibodies), an ELISA was developed using carbamylated FCS (Ca-FCS) and non-modified FCS as antigens. Analyzing sera of 40 RA patients and 40 controls, it was observed that sera of RA patients reacted with Ca-FCS as compared to sera obtained from healthy subjects with both IgG (FIG. 7, Panels A and B) and IgA (FIG. 7, Panels C and D) reactivity. The enhanced reactivity of RA sera to Ca-FCS is further emphasized after subtraction of the reactivity against unmodified FCS (FIG. 7, Panels C and E). Since citrulline and homocitrulline are two rather similar amino acids (FIG. 6), it was next determined whether ACPA also recognizes homocitrulline when located at the same position as citrulline in a peptide. For this purpose, ELISAs were performed using a citrullinated Fib peptide known to be recognized by ACPA.⁽²⁰⁾ Within this peptide backbone, a citrulline, an arginine, a homocitrulline or a lysine residue was introduced for further analysis. Analyzing a set of 76 RA sera, it was observed that ACPA only recognized the citrullinated peptide, but not the arginine-containing or the homocitrulline-containing peptide (FIG. 8, Panel A). These data indicate that ACPA can discriminate between citrulline and homocitrulline present within the same peptide backbone. Next, analysis was performed as to whether there is cross-reactivity between anti-CarP antibodies and ACPA for binding to post-translationally modified proteins. Therefore, inhibition studies were performed using sera that were reactive to both citrullinated and carbamylated antigens. The binding of anti-CarP antibodies to Ca-FCS-coated plates following pre-incubation with Ca-FCS, citrullinated FCS (Ci-FCS), native FCS or by citrullinated peptides used to detect ACPA (CCP1) was analyzed. Following pre-incubation, it was observed that anti-CarP antibody binding to Ca-FCS can only be inhibited by Ca-FCS but not by citrullinated FCS (Ci-FCS), native FCS or by peptides used to detect ACPA (FIG. 8, Panel B). The reverse inhibition experiment was performed where the binding of ACPA to plates coated with Ci-FCS following the same pre-incubation procedure was analyzed. It was observed that ACPA binding to Ci-FCS could only be inhibited by Ci-FCS and the citrullinated peptide but not by Ca-FCS, non-modified FCS, or the arginine form of the peptide (FIG. 8, Panel C). Together, these data indicate that anti-CarP antibodies and ACPA are not (or only limited) cross-reactive and specifically directed against homocitrulline, respectively, citrulline-containing antigens. Since all observations described above were made using ELISA, confirmation of the findings using a different technique was desirable. For this reason, a Western blot analysis was performed using FCS, Ca-FCS and Ci-FCS on reduced gels, followed by Western blotting. The different blots were incubated with sera of individuals that were either anti-CarP positive and ACPA negative or anti-CarP negative and ACPA positive. A positive staining of the anti-CarP-positive sample was observed only on Ca-FCS but not on Ci-FCS or FCS (FIG. 8, Panel D). In contrast, the anti-CarP negative, ACPA-positive sample reacted to Ci-FCS, but not to Ca-FCS and FCS (FIG. 8, Panel D). To confirm the presence of anti-CarP antibodies, these experiments were repeated using a more defined protein, human Fib, as target antigen. Fib was citrullinated by PAD (Ci-Fib) or carbamylated by cyanate (Ca-Fib). The non-modified form (Fib), Ci-Fib and Ca-Fib were used as antigens in ELISA. Similar to the observations for FCS, significant binding of antibodies to the Ci-Fib and the Ca-Fib but not to the Fib-coated wells was observed (FIG. 9, Panel A). This was largely restricted to the RA sera and not the controls (p=<0.0001). To analyze cross-reactivity, inhibition studies were performed as described above. ELISA analyses confirmed that ACPA and anti-CarP antibodies are largely non-cross-reactive. To ensure that reactivity toward carbamylated proteins is mediated by the antigen-binding-part of the antibodies, F(ab′)2 was generated. As expected, F(ab′)2, generated from anti-CarP IgG-positive samples but not from negative samples, display anti-CarP reactivity (FIG. 9, Panels C and D). As observed using intact antibodies, F(ab′)2 reactivity toward Ca-Fib could be inhibited specifically by Ca-Fib, whereas F(ab′)2 reactivity toward Ci-Fib could only be inhibited specifically by Ci-Fib (FIG. 9, Panel E).

Collectively, these data indicate that anti-CarP antibodies and ACPA recognize different antigens, one recognizing citrullinated proteins (ACPA) and the other carbamylated proteins (anti-CarP). Likewise, these data indicate that antigen recognition is most likely mediated via the variable domains present in the F(ab′)2 fragments.

Anti-CarP Antibodies are Present in RA

Following the identification of anti-CarP antibodies as an autoantibody family separate from ACPA, quantifying the presence of these anti-CarP antibodies in a large population of RA patients and controls was desired. For this reason, first, a standard was generated comprising a pool of anti-CarP antibody-positive sera. This standard displayed a specific, dose-dependent, binding of both IgG and IgA to carbamylated FCS (Ca-FCS) but no binding to unmodified FCS (FIG. 10, Panels A and B). For this analysis, the FCS-based assay was again used in an attempt to capture as many anti-CarP reactivities as possible. A cut-off for positivity was established using sera of 305 healthy individuals as described in the methods section. Using this approach, it was observed that 45% of the sera of RA patients analyzed are positive for IgG anti-CarP antibodies (FIG. 10, Panel C). Likewise, 43% of sera from RA patients tested are positive for IgA anti-CarP-antibodies (FIG. 10, Panel D).

Anti-CarP Antibodies are Also Present in Sera of Anti-CCP2-Negative RA Patients

The group of RA patients analyzed in this study consisted of both ACPA-positive and ACPA-negative individuals, as measured by the CCP2 assay. Therefore, the association between anti-CarP antibodies and anti-CCP2 antibodies was analyzed next. The presence of anti-CarP antibodies and anti-CCP2 antibodies showed a limited degree of correlation when analyzing the entire RA population (r²=0.27, p<0.001 for anti-CarP IgG or r²=0.15, p<0.001 for IgA). However, substantial numbers of RA patients were also identified that are only positive for anti-CCP2 antibodies as well as a group of patients that is only positive for anti-CarP antibodies (FIG. 10, Panels E and F). It was observed that approximately 16% of the anti-CCP2-negative RA patients displayed anti-CarP IgG antibodies, whereas 30% of the anti-CCP2-negative RA patients tested positive for anti-CarP IgA (FIG. 10, Panels G and H). These data indicate that the presence of anti-CarP antibodies overlaps with the occurrence of anti-CCP2 antibodies, but that this overlap is not absolute as over 30% of the anti-CCP2-negative patients harbor anti-CarP antibodies. In total, more than 35% of all anti-CCP2-negative patients have either anti-CarP IgG or IgA antibodies.

Anti-CarP Antibodies are Associated with More Severe Radiological Damage

The presence of ACPA is associated with a more severe clinical disease course as measured by radiological damage. To analyze whether the presence of anti-CarP antibodies are also predictive for a more severe disease course, the extent of joint damage over time between anti-CarP-positive and -negative patients participating in the Leiden EAC cohort was compared. This cohort is an inception cohort of patients with recent-onset arthritis where X-rays of hands and feet are taken of all RA patients at yearly intervals to assess radiological damage using the Sharp/van der Heijde method.⁽²¹⁾ It was observed that the presence of anti-CarP IgG strongly associates with a more severe disease progression. Patients positive for anti-CarP IgG had more joint destruction over seven years than IgG-negative patients without (β=2.01, 95% CI 1.68-2.40, p=8.68×10⁻¹⁴) or with correction of ACPA and RF (β=1.41, 95% CI 1.13-1.76, p=0.002) (FIG. 13). Anti-CarP IgA was associated with more joint destruction over seven years than anti-CarP IgA-negative patients without correction of ACPA and RF (β=1.21, 95% CI 1.01-1.45, p=0.041) but not after correction (p=0.855) (FIG. 13). As the analysis described above does not show whether anti-CarP antibodies predict radiological progression in the anti-CCP2-negative, anti-CCP2-positive or both RA subgroups, a stratified analysis was next performed. Importantly, this analysis revealed that the presence of anti-CarP IgG is associated with a more severe joint damage in the anti-CCP2-negative subgroup (β=1.86, 95% CI 1.41-2.66, p=1.8×10⁻⁵) (FIG. 11). Likewise, a similar trend toward more joint damage over time was observed for anti-CCP2-negative patients tested positive for IgA anti-CarP antibodies (β=1.25, 95% CI 0.98-1.58, p=0.071) (FIG. 13). In contrast, in the anti-CCP2-positive subgroup, which is already characterized by severe joint destruction, no additional increase was observed in individuals that also harbored anti-CarP antibodies (FIG. 13). Together, these data indicate that the detection of anti-CarP antibodies at baseline is predictive for a more destructive disease course in anti-CCP2-negative RA as measured by the Sharp/van der Heijde method.

Discussion

A family of autoantibodies that recognize carbamylated proteins, anti-CarP antibodies can be detected in sera of RA patients. Both inhibition studies and cohort studies show that anti-CarP antibodies and ACPA represent two different and independent autoantibody families, one recognizing carbamylated proteins and the other citrullinated proteins. The data show that anti-CarP antibodies and ACPA are, by and large, non-cross-reactive, although no exclusion is made that some cross-reactivity exists at the population level as is also indicated in recent data obtained in rabbits after vaccination with carbamylated proteins.⁽¹⁴⁾ Interestingly, positivity for anti-CarP antibodies is related to clinical outcome as individuals positive for anti-CarP IgG, but negative for anti-CCP2 antibodies, have a more destructive disease course as compared to anti-CarP IgG-negative RA patients.

It is currently unknown which proteins undergo post-translational modifications like carbamylation. Carbamylation is mediated by cyanate, which is in equilibrium with urea. Increased urea concentrations, smoking and inflammation have been reported to shift this equilibrium toward cyanate and, hence, enhanced carbamylation.⁽¹³⁾ Since currently no in vivo-relevant targets for anti-CarP antibodies are known, a complex protein mixture was used as an initial source of carbamylated protein antigens for the detection of anti-CarP antibodies. Western blot analyses indicate the recognition by anti-CarP antibodies of at least one dominant protein present in FCS after carbamylation employing cyanate (representing high urea concentrations) (FIG. 8, Panel D). However, these data are likely not to represent the in vivo situation where carbamylation is a more gradual, but constantly occurring process.⁽²²⁾ In this respect, it is likely that especially long-lived proteins acquire homocitrulline residues over time as carbamylation is nearly irreversible and, thus, will lead to the accumulation of homocitrulline residues on proteins with a long half-life. Intriguingly, the joint is known for the presence of long-lived proteins such as collagens and other cartilage-expressed proteins. Therefore, it is conceivable that such matrix-proteins will accumulate homocitrulline residues during life, especially under conditions of inflammation. Indeed, it has been shown that homocitrulline is present in the joint,⁽¹¹⁾ possibly representing the long-lived nature of many joint-derived proteins. It will be interesting to know the identity of these proteins and whether these can serve as a target for anti-CarP antibodies.

The molecular nature of the antigens recognized by ACPA was identified more than 15 years ago by describing that citrulline is an essential constituent of antigens recognized by these RA-specific antibodies.^((23, 24)) This finding has made considerable impact as it has opened up the way to relevant and novel insights into RA diagnosis and etiopathology.⁽¹⁾ For example, ACPA are now part of the new ACR/EULAR criteria for RA,⁽²⁵⁾ and have been implicated in RA pathogenesis, both in animal models^((26, 27, 28)) and in ex vivo human studies.^((29, 30, 31, 32)) Importantly, the description of ACPA has led to the realization that RA constitutes at least two clinical syndromes that share many clinical features, but differ with respect to genetic background, predisposing environmental factors and clinical progression/remission.^((33, 3, 4, 34, 35)) Although it is clearly too early to allow any firm conclusions, it is tempting to speculate that anti-CarP antibodies also contribute to disease pathogenesis and/or display diagnostic value, given the similar nature of the antigens recognized and their presence in ACPA-negative disease.

The presence of anti-CarP antibodies in anti-CCP2-negative disease is highly intriguing as it could potentially represent a novel biomarker that positively identifies at least part of this manifestation of RA. To gain further insight into this possibility, it is important to establish whether the presence of anti-CarP antibodies is specific for RA or also found in other rheumatic diseases, as well as whether their presence predict the development of (ACPA-negative) RA in patients suffering from early unclassified RA and/or joint complaints such as arthralgia.

To establish a cut-off to define a positive sample, the presence of IgG and IgA directed against Ca-FCS and FCS in sera of healthy controls was analyzed. All samples were tested for reactivity toward Ca-FCS and FCS, and absorbance values were converted into aU/mL using an anti-CarP antibody-positive standard present on the same plate. Since sera from several individual subjects also displayed reactivity toward non-modified FCS, the “FCS reactivity” was subtracted from the reactivity toward Ca-FCS using aU/mL as defined by the standard curve. Subsequently, the cut-off was calculated as the mean plus two times standard deviation and applied the cut-off to the data of the RA patients following a similar strategy. The disadvantage of this method is that a standard is used on Ca-FCS for the determination of aU/mL toward FCS, another antigenic entity. However, this method did allow the calculation of a specific response to the post-translational modification.

Every method of establishing a cut-off has advantages and limitations. Therefore, the observations were subsequently confirmed using another strategy as well, by calculating the cut-off as the mean plus two times standard deviation of the anti-Ca-FCS response in controls. This cut-off was applied to the data of the RA patients as was also employed before.⁽³⁶⁾ The association with radiological progression of IgG in ACPA-negative RA remains significant, albeit with a lower level of significance (p=0.001).

From a clinical perspective, the detection of anti-CarP antibodies in early arthritis could be highly rewarding since they predict a more severe disease course. Since early aggressive treatment in RA has been shown to prevent future damage,^((37, 38)) the detection of anti-CarP antibodies might be beneficial to identify anti-CCP2-negative patients at risk to develop severe disease. The identification of such patients might be important to guide treatment decisions early after onset of symptoms, especially in early arthritis patients that are difficult to classify.

In conclusion, in addition to the autoantibody system that recognizes citrullinated proteins (ACPA), an autoantibody system against carbamylated proteins (anti-CarP) is present in sera of RA patients. Detection of anti-CarP antibodies could offer new possibilities to identify patients at risk for a severe disease course.

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TABLE I List of lysine-containing peptides of fibrinogen alpha and their homocitrulline-containing  counterparts Lysine-containing peptides  of fibrinogen alpha Homocitrulline-containing peptides of fibrinogen alpha SEQ ID SEQ ID NO: NO: 1 4 RVVERHQSACKDSDWPFCSDE 1 5 RVVERHQSAChomocitDSDWPFCSDE 2 6 PFCSDEDWNYKCPSGCRMKGL 2 7 PFCSDEDWNYhomocitCPSGCRMhomocitGL 3 8 NYKCPSGCRMKGLIDEVNQDF 3 9 NYhomocitCPSGCRMhomocitGLIDEVNQDF 4 10 VNQDFTNRINKLKNSLFEYQK 4 11 VNQDFTNRINhomocitLhomocitNSLFEYQhomocit 5 12 QDFTNRINKLKNSLFEYQKNN 5 13 QDFTNRINhomocitLhomocitNSLFEYQhomocitNN 6 14 KLKNSLFEYQKNNKDSHSLTT 6 15 homocitLhomocitNSLFEYQhomocitNNhomocitDSHSLTT 7 16 NSLFEYQKNNKDSHSLTTNIM 7 17 NSLFEYQhomocitNNhomocitDSHSLTTNIM 8 18 EDLRSRIEVLKRKVIEKVQHI 8 19 EDLRSRIEVLhomocitRhomocitVIEhomocitVQHI 9 20 LRSRIEVLKRKVIEKVQHIQL 9 21 LRSRIEVLhomocitRhomocitVIEhomocitVQHIQL 10 22 IEVLKRKVIEKVQHIQLLQKN 10 23 IEVLhomocitRhomocitVIEhomocitVQHIQLLQhomocitN 11 24 EKVQHIQLLQKNVRAQLVDMK 11 25 EhomocitVQHIQLLQhomocitNVRAQLVDMhomocit 12 26 KNVRAQLVDMKRLEVDIDIKI 12 27 homocitNVRAQLVDMhomocitRLEVDIDIhomocitI 13 28 MKRLEVDIDIKIRSCRGSCSR 13 29 MhomocitRLEVDIDIhomocitIRSCRGSCSR 14 30 SRALAREVDLKDYEDQQKQLE 14 31 SRALAREVDLhomocitDYEDQQhomocitQLE 15 32 VDLKDYEDQQKQLEQVIAKDL 15 33 VDLhomocitDYEDQQhomocitQLEQVIAhomocitDL 16 34 QQKQLEQVIAKDLLPSRDRQH 16 35 QQhomocitQLEQVIAhomocitDLLPSRDRQH 17 36 SRDRQHLPLIKMKPVPDLVPG 17 37 SRDRQHLPLIhomocitMhomocitPVPDLVPG 18 38 DRQHLPLIKMKPVPDLVPGNF 18 39 DRQHLPLIhomocitMhomocitPVPDLVPGNF 19 40 PVPDLVPGNFKSQLQKVPPEW 19 41 PVPDLVPGNFhomocitSQLQhomocitVPPEW 20 42 VPGNFKSQLQKVPPEWKALTD 20 43 VPGNFhomocitSQLQhomocitVPPEWhomocitALTD 21 44 SQLQKVPPEWKALTDMPQMRM 21 45 SQLQhomocitVPPEWhomocitALTDMPQMRM 22 46 SSGTGGTATWKPGSSGPGSTG 22 47 SSGTGGTATWhomocitPGSSGPGSTG 23 48 PGTRREYHTEKLVTSKGDKEL 23 49 PGTRREYHTEhomocitLVTShomocitGDhomocitEL 24 50 EYHTEKLVTSKGDKELRTGKE 24 51 EYHTEhomocitLVTShomocitGDhomocitELRTGhomocitE 25 52 TEKLVTSKGDKELRTGKEKVT 25 53 TEhomocitLVTShomocitGDhomocitELRTGhomocitEhomocitVT 26 54 SKGDKELRTGKEKVTSGSTTT 26 55 ShomocitGDhomocitELRTGhomocitEhomocitVTSGSTTT 27 56 GDKELRTGKEKVTSGSTTTTR 27 57 GDhomocitELRTGhomocitEhomocitVTSGSTTTTR 28 58 STTTTRRSCSKTVTKTVIGPD 28 59 STTTTRRSCShomocitTVThomocitTVIGPD 29 60 TRRSCSKTVTKTVIGPDGHKE 29 61 TRRSCShomocitTVThomocitTVIGPDGHhomocitE 30 62 TKTVIGPDGHKEVTKEVVTSE 30 63 ThomocitTVIGPDGHhomocitEVThomocitEVVTSE 31 64 IGPDGHKEVTKEVVTSEDGSD 31 65 IGPDGHhomocitEVThomocitEVVTSEDGSD 32 66 AAFFDTASTGKTFPGFFSPML 32 67 AAFFDTASTGhomocitTFPGFFSPML 33 68 GSESGIFTNTKESSSHHPGIA 33 69 GSESGIFTNThomocitESSSHHPGIA 34 70 PGIAEFPSRGKSSSYSKQFTS 34 71 PGIAEFPSRGhomocitSSSYShomocitQFTS 35 72 PSRGKSSSYSKQFTSSTSYNR 35 73 PSRGhomocitSSSYShomocitQFTSSTSYNR 36 74 YNRGDSTFESKSYKMADEAGS 36 75 YNRGDSTFEShomocitSYhomocitMADEAGS 37 76 GDSTFESKSYKMADEAGSEAD 37 77 GDSTFEShomocitSYhomocitMADEAGSEAD 38 78 EADHEGTHSTKRGHAKSRPVR 38 79 EADHEGTHSThomocitRGHAhomocitSRPVR 39 80 GTHSTKRGHAKSRPVRDCDDV 39 81 GTHSThomocitRGHAhomocitSRPVRDCDDV 40 82 SGTQSGIFNIKLPGSSKIFSV 40 83 SGTQSGIFNIhomocitLPGSShomocitIFSV 41 84 IFNIKLPGSSKIFSVYCDQET 41 85 IFNIhomocitLPGSShomocitIFSVYCDQET 42 86 LNFNRTWQDYKRGFGSLNDEG 42 87 LNFNRTWQDYhomocitRGFGSLNDEG 43 88 VRGIHTSPLGKPSLSP 43 89 VRGIHTSPLGhomocitPSLSP

TABLE II List of lysine-containing peptides of fibrinogen beta and their homocitrulline-containing counterparts Lysine-containing peptides of  fibrinogen beta Homocitrulline-containing peptides of fibrinogen beta SEQ ID SEQ ID NO: NO: 1 90 MKRMVSWSFHKL 1 91 MhomocitRMVSWSFHhomocitL 2 92 MKRMVSWSFHKLKTMKHLLLL 2 93 MhomocitRMVSWSFHhomocitLhomocitTMhomocitHLLLL 3 94 RMVSWSFHKLKTMKHLLLLLL 3 95 RMVSWSFHhomocitLhomocitTMhomocitHLLLLLL 4 96 SWSFHKLKTMKHLLLLLLCVF 4 97 SWSFHhomocitLhomocitTMhomocitHLLLLLLCVF 5 98 LLLLLCVFLVKSQGVNDNEEG 5 99 LLLLLCVFLVhomocitSQGVNDNEEG 6 100 FSARGHRPLDKKREEAPSLRP 6 101 FSARGHRPLDhomocithomocitREEAPSLRP 7 102 SARGHRPLDKKREEAPSLRPA 7 103 SARGHRPLDhomocithomocitREEAPSLRPA 8 104 SGGGYRARPAKAAATQKKVER 8 105 SGGGYRARPAhomocitAAATQhomocithomocitVER 9 106 ARPAKAAATQKKVERKAPDAG 9 107 ARPAhomocitAAATQhomocithomocitVERhomocitAPDAG 10 108 RPAKAAATQKKVERKAPDAGG 10 109 RPAhomocitAAATQhomocithomocitVERhomocitAPDAGG 11 110 AAATQKKVERKAPDAGGCLHA 11 111 AAATQhomocithomocitVERhomocitAPDAGGCLHA 12 112 SSSFQYMYLLKDLWQKRQKQV 12 113 SSSFQYMYLLhomocitDLWQhomocitRQhomocitQV 13 114 YMYLLKDLWQKRQKQVKDNEN 13 115 YMYLLhomocitDLWQhomocitRQhomocitQVhomocitDNEN 14 116 LLKDLWQKRQKQVKDNENVVN 14 117 LLhomocitDLWQhomocitRQhomocitQVhomocitDNENVVN 15 118 DLWQKRQKQVKDNENVVNEYS 15 119 DLWQhomocitRQhomocitQVhomocitDNENVVNEYS 20 120 PVVSCEEIIRKGGETSEMYLI 20 121 PVVSCEEIIRhomocitGGETSEMYLI 21 122 MYLIQPDSSVKPYRVYCDMNT 21 123 MYLIQPDSSVhomocitPYRVYCDMNT 22 124 VDFGRKWDPYKQGFGNVATNT 22 125 VDFGRhomocitWDPYhomocitQGFGNVATNT 23 126 FGNVATNTDGKNYCGLPGEYW 23 127 FGNVATNTDGhomocitNYCGLPGEYW 24 128 LPGEYWLGNDKISQLTRMGPT 24 129 LPGEYWLGNDhomocitISQLTRMGPT 25 130 TELLIEMEDWKGDKVKAHYGG 25 131 TELLIEMEDWhomocitGDhomocitVhomocitAHYGG 26 132 LIEMEDWKGDKVKAHYGGFTV 26 133 LIEMEDWhomocitGDhomocitVhomocitAHYGGFTV 27 134 EMEDWKGDKVKAHYGGFTVQN 27 135 EMEDWhomocitGDhomocitVhomocitAHYGGFTVQN 28 136 GGFTVQNEANKYQISVNKYRG 28 137 GGFTVQNEANhomocitYQISVNhomocitYRG 29 138 EANKYQISVNKYRGTAGNALM 29 139 EANhomocitYQISVNhomocitYRGTAGNALM 30 140 NDGWLTSDPRKQCSKEDGGGW 30 141 NDGWLTSDPRhomocitQCShomocitEDGGGW 31 142 LTSDPRKQCSKEDGGGWWYNR 31 143 LTSDPRhomocitQCShomocitEDGGGWWYNR 32 144 WGGQYTWDMAKHGTDDGVVWM 32 145 WGGQYTWDMAhomocitHGTDDGVVWM 33 146 TDDGVVWMNWKGSWYSMRKMS 33 147 TDDGVVWMNWhomocitGSWYSMRhomocitMS 34 148 NWKGSWYSMRKMSMKIRPFFQ 34 149 NWhomocitGSWYSMRhomocitMSMhomocitIRPFFQ 35 150 SWYSMRKMSMKIRPFFPQQ 35 151 SWYSMRhomocitMSMhomocitIRPFFPQQ

TABLE III List of lysine-containing peptides of fibrinogen gamma and their homocitrulline- containing counterparts Lysine-containing peptides of   fibrinogen gamma Homocitrulline-containing peptides of fibrinogen gamma SEQ ID SEQ ID NO: NO: 1 152 IADFLSTYQTKVDKDLQSLED 1 153 IADFLSTYQThomocitVDhomocitDLQSLED 2 154 FLSTYQTKVDKDLQSLEDILH 2 155 FLSTYQThomocitVDhomocitDLQSLEDILH 3 156 LEDILHQVENKTSEVKQLIKA 3 157 LEDILHQVENhomocitTSEVhomocitQLIhomocitA 4 158 HQVENKTSEVKQLIKAIQLTY 4 159 HQVENhomocitTSEVhomocitQLIhomocitAIQLTY 5 160 NKTSEVKQLIKAIQLTYNPDE 5 161 NhomocitTSEVhomocitQLIhomocitAIQLTYNPDE 6 162 QLTYNPDESSKPNMIDAATLK 6 163 QLTYNPDESShomocitPNMIDAATLhomocit 7 164 KPNMIDAATLKSRKMLEEIMK 7 165 homocitPNMIDAATLhomocitSRhomocitMLEEIMhomocit 8 166 MIDAATLKSRKMLEEIMKYEA 8 167 MIDAATLhomocitSRhomocitMLEEIMhomocitYEA 9 168 KSRKMLEEIMKYEASILTHDS 9 169 homocitSRhomocitMLEEIMhomocitYEASILTHDS 10 170 LQEIYNSNNQKIVNLKEKVAQ 10 171 LQEIYNSNNQhomocitIVNLhomocitEhomocitVAQ 11 172 NSNNQKIVNLKEKVAQLEAQC 11 173 NSNNQhomocitIVNLhomocitEhomocitVAQLEAQC 12 174 NNQKIVNLKEKVAQLEAQCQE 12 175 NNQhomocitIVNLhomocitEhomocitVAQLEAQCQE 13 176 QLEAQCQEPCKDTVQIHDITG 13 177 QLEAQCQEPChomocitDTVQIHDITG 14 178 DTVQIHDITGKDCQDIANKGA 14 179 DTVQIHDITGhomocitDCQDIANhomocitGA 15 180 TGKDCQDIANKGAKQSGLYFI 15 181 TGhomocitDCQDIANhomocitGAhomocitQSGLYFI 16 182 DCQDIANKGAKQSGLYFIKPL 16 183 DCQDIANhomocitGAhomocitQSGLYFIhomocitPL 17 184 GAKQSGLYFIKPLKANQQFLV 17 185 GAhomocitQSGLYFIhomocitPLhomocitANQQFLV 18 186 GSGNGWTVFQKRLDGSVDFKK 18 187 GSGNGWTVFQhomocitRLDGSVDFhomocithomocit 19 188 QKRLDGSVDFKKNWIQYKEGF 19 189 QhomocitRLDGSVDFhomocithomocitNWIQYhomocitEGF 20 190 KRLDGSVDFKKNWIQYKEGFG 20 191 homocitRLDGSVDFhomocithomocitNWIQYhomocitEGFG 21 192 VDFKKNWIQYKEGFGHLSPTG 21 193 VDFhomocithomocitNWIQYhomocitEGFGHLSPTG 22 194 GTTEFWLGNEKIHLISTQSAI 22 195 GTTEFWLGNEhomocitIHLISTQSAI 23 196 RTSTADYAMFKVGPEADKYRL 23 197 RTSTADYAMFhomocitVGPEADhomocitYRL 24 198 AMFKVGPEADKYRLTYAYFAG 24 199 AMFhomocitVGPEADhomocitYRLTYAYFAG 25 200 GFDFGDDPSDKFFTSHNGMQF 25 201 GFDFGDDPSDhomocitFFTSHNGMQF 26 202 QFSTWDNDNDKFEGNCAEQDG 26 203 QFSTWDNDNDhomocitFEGNCAEQDG 27 204 EQDGSGWWMNKCHAGHLNGVY 27 205 EQDGSGWWMNhomocitCHAGHLNGVY 28 206 GVYYQGGTYSKASTPNGYDNG 28 207 GVYYQGGTYShomocitASTPNGYDNG 29 208 YDNGIIWATWKTRWYSMKKTT 29 209 YDNGIIWATWhomocitTRWYSMhomocithomocitTT 30 210 ATWKTRWYSMKKTTMKIIPFN 30 211 ATWhomocitTRWYSMhomocithomocitTTMhomocitIIPFN 31 212 TWKTRWYSMKKTTMKIIPFNR 31 213 TWhomocitTRWYSMhomocithomocitTTMhomocitIIPFNR 32 214 RWYSMKKTTMKIIPFNRLTIG 32 215 RWYSMhomocithomocitTTMhomocitIIPFNRLTIG 

The invention claimed is:
 1. A kit for the detection of anti-Ca-Fib antibodies, in a body fluid of an individual, the kit comprising: an in vitro carbamylated fibrinogen peptide comprising a peptide selected from the group consisting of the odd numbered SEQ ID NOs:5-215, a solid support, and an anti-human immunoglobulin antibody.
 2. A kit for detecting anti-CarP antibodies in a body fluid of an individual, the kit comprising: an in vitro carbamylated fetal calf serum (FCS), a solid support, and an anti-human immunoglobulin antibody.
 3. The kit of claim 2, wherein the anti-human immunoglobulin antibody comprises an anti-human IgG antibody.
 4. The kit of claim 2, wherein the anti-human immunoglobulin antibody comprises an anti-human IgA antibody.
 5. The kit of claim 2 further comprising: a citrullinated protein or peptide.
 6. The kit of claim 1 comprising two carbamylated fibrinogen proteins or peptides.
 7. The kit of claim 6 comprising three carbamylated proteins or peptides.
 8. The kit of claim 1, further comprising a fibrinogen peptide having a sequence selected from the group consisting of the even numbered SEQ ID NOS:4-214.
 9. The kit of claim 2, further comprising a non-modified FCS or a citrullinated FCS. 